Significant biological interest has surrounded the transcription factor NF-kappaB for the following reasons: (i) its inducibility by cytokines and other immunological/inflammatory mediators, (ii) its interesting regulation through interactions with the IkappaB inhibitory proteins, and (iii) its critical involvement with a range of diseases (both inflammatory and oncological, Baldwin, J. Clin. Inv. 107, 3-6). Studies on NF-kappaB regulation have led to a fairly cohesive model which involves: the activation of the IkappaB kinase (IKK, containing IKKalpha, IKKbeta, IKKgamma), phosphorylation of IkappaB by IKK, IkappaB degradation, and nuclear accumulation of NF-kappaB. In this regard, one of the IKK subunits (IKKbeta) is required for TNF induction of NF-kappaB through its ability to control IkappaB phosphorylation. The lack of involvement of IKKalpha in this pathway has led to the proposal that this IKK subunit is not involved in the cytokine controlled NF-kappaB( regulatory pathway. However, emerging data has revealed a more complex and significantly more interesting view of NF-kappaB regulation and IKK function. Thus, it has been shown that IKKalpha is required for the induction of NF-kappaB-dependent gene expression downstream of TNF-induced signaling and that GSK-3beta (the kinase involved in Wnt signaling) controls NF-kappaB functional activity downstream of IkappaB degradation and NF-kappaB nuclear accumulation. These results indicate that regulation of NF-kappaB transcriptional competence is controlled at the nuclear level in addition to mechanisms which regulate release from IkappaB. Additionally, IKKalpha (but IKKbeta) is required for TLbetaR-induced signaling controlling NF-kappaB2 activation and the control of specific chemokine/cytokine gene expression. Further evidence for functional differences between IKKalpha and IKKbeta is provided by the phenotype of the IKKalpha null animal which exhibits skin and skeletal abnormalities associated with a block on keratinocyte differentiation that may be independent of NF-kappaB. Our preliminary data demonstrate new findings which are likely to explain major mechanisms associated with the regulation of NF-kappaB-dependent, and potentially NF-kappaB-independent, gene expression. Thus, we have found that IKKalpha is induced to accumulate in the nucleus following cytokine treatment of a variety of cells. Surprisingly, we find that IKKalpha and IKKbeta are both induced to associate with the promoter regions of the NF-kappaB-regulated genes. Evidence is presented that IKKalpha controls cytokine inducible histone H3 ser10 phosphorylation, an initiating event in the control of histone modification described by the 'histone code hypothesis.' Furthermore, we find that IKKalpha is required to control epidermal growth factor (EGF)-induced histone H3 ser10 phosphorylation and that GSK-3beta controls the phosphorylation of the p65 subunit on ser536. We propose to analyze the structural and regulatory features of IKKalpha and IKKbeta involved in modulating chromatin function extending the hypothesis to the existence of separate IKKalpha and IKKbeta-containing complexes regulating chromatin modification. We also hypothesize that IKKalpha is involved in the control of chromatin modification for genes that are not NF-kappaB-dependent, potentially explaining the requirement of IKKalpha in controlling keratinocyte differentiation. Finally, we propose to determine the role of GSK-3beta in controlling p65 ser56 phosphorylation, relating this to the potential involvement of the IKKs. Additionally, using restoration experiments in p65 null cells and knock-in technology we propose to analyze the requirement of p65 ser536 phosphorylation in overall NF-kappaB function. These experiments have the potential to provide new, significant insight into nuclear mechanisms involved in controlling NF-kappaB-dependent (and possibly independent) gene expression through control of chromatin modification and assembly of active transcription complexes.